Antibody Targeting Through a Modular Recognition Domain

ABSTRACT

Antibodies containing one or more modular recognition domains (MRDs) used to target the antibodies to specific sites are described. The use of the antibodies containing one or more modular recognition domains to treat disease, and methods of making antibodies containing one or more modular recognition domains are also described.

FIELD OF THE INVENTION

This invention relates generally to antibodies containing one or moremodular recognition domains and more specifically to the use of theantibodies containing one or more modular recognition domains to treatdisease, and methods of making antibodies containing one or more modularrecognition domains.

BACKGROUND

Catalytically active monoclonal antibodies (Abs) can be used forselective prodrug activation and chemical transformations. MonoclonalAbs with aldolase activity have emerged as highly efficient catalystsfor a number of chemical transformations, particularly aldol andretro-aldol reactions. The retro-aldolase activity of Abs, such as 38C2and 93F3, have allowed researchers to design, synthesize, and evaluateprodrugs of various chemotherapeutic agents that can be activated byretro-aldol reactions. (Construction of 38C2 was described in WO97/21803, herein incorporated by reference). 38C2 contains an antibodycombining site that catalyzes the aldol addition reaction between analiphatic donor and an aldehyde acceptor. In a syngeneic mouse model ofneuroblastoma, systemic administration of an etoposide prodrug andintra-tumor injection of 38C2 inhibited tumor growth.

One drawback in the use of catalytic Abs is that they lack a device totarget the catalytic Ab to the malignant cells. Previous studiesdemonstrated that in an antibody-directed enzyme prodrug therapy (ADEPT)or antibody-directed abzyme prodrug therapy (ADAPT) approach, enzymes orcatalytic antibodies can be directed to tumor cells by chemicalconjugation or recombinant fusion to targeting antibodies. However, amore efficient alternative would be using the catalytic antibody fusedto a targeting peptide located outside the antibody combining site,thereby leaving the active site available for the prodrug activation.For example, the fusion of Ab 38C2 to an integrin αvβ3-binding peptidewould selectively localize the antibody to the tumor and/or the tumorvasculature and trigger prodrug activation at that site. The potentialtherapy of this approach is supported by preclinical and phase Illclinical data suggesting that peptides can be converted into viabledrugs through fusion to antibody Fc regions.

The development of bispecific or multi-specific antibodies that targettwo or more cancer targets simultaneously and or activate prodrugsoffers a novel and promising solution to attacking cancer and otherdiseases. Such antibodies are exemplified in FIG. 1 of the presentapplication. Studies of bispecific antibodies (BsAb) that simultaneouslytarget two tumor-associated antigens (e.g. growth factor receptors) fordown-regulation of multiple cell proliferation/survival pathways hasprovided support for this approach. Traditionally, bispecific antibodieshave been prepared by chemically linking two different monoclonalantibodies or by fusing two hybridoma cell lines to produce ahybrid-hybridoma. Dual-specific, tetravalent IgG-like molecules, ordual-variable-domain immunoglobins, have been engineered from twomonoclonal antibodies. These dual-variable-domain immunoglobins arecapable of binding both antigens in the presence of serum. However,these approaches present challenges with respect to manufacturing, yieldand purity.

A variety of recombinant methods have been developed for efficientproduction of small BsAb fragments such as diabody, minibody, andFab-scFv fusion proteins. These BsAb fragments may possess someadvantages over the full-length IgG-like molecules for certain clinicalapplications, such as for tumor radio-imaging and targeting, because ofbetter tissue penetration and faster clearance from the circulation. Onthe other hand, IgG-like BsAb may prove to be preferred over smallerBsAb fragments for other in vivo applications, specifically for oncologyindications, by providing the Fc domain that confers long serumhalf-life and supports secondary immune function, such asantibody-dependent cellular cytotoxicity and complement-mediatedcytotoxicity. Unlike their fragment counterparts, engineering andproduction of recombinant IgG-like BsAb has been, however, rathertechnically challenging due to their large size (˜150-200 kDa) andstructural complexity. Success in the field, as judged by successfulapplication in animal models, has been very limited. Recently, with theexamination of a variety of constructs, the efficient expression of Fcdomain containing BsAb molecules in mammalian cells has made somestrides.

Another approach that has been used to target antibodies is through theuse of peptibodies. Peptibodies are essentially peptide fusions withantibody Fc regions. Given the success of studies using random peptidelibraries to find high-affinity peptide ligands for a wide variety oftargets, fusion of such peptides to antibody Fc regions provides a meansof making peptides into therapeutic candidates by increasing theircirculatory half-life and activity through increased valency.

Protein interactions with other molecules is basic to biochemistry.Protein interactions include receptor-ligand interactions,antibody-antigen interactions, cell-cell contact and pathogeninteractions with target tissues. Protein interactions can involvecontact with other proteins, with carbohydrates, oligosaccharides,lipids, metal ions and the like materials. The basic unit of proteininteraction is the region of the protein involved in contact andrecognition, and is referred to as the binding site or target site.

Peptides derived from phage display libraries typically retain theirbinding characteristics when linked to other molecules. Specificpeptides of this type can be treated as modular specificity blocks ormolecular recognition domains (MRDs) that can be combined to create asingle protein with binding specificities for several defined targets.

An example of a such a defined target site is integrin. Integrins are afamily of transmembrane cell adhesion receptors that are composed of αand β subunits and mediate cell attachment to proteins within theextracellular matrix. At present, eighteen α and eight β subunits areknown; these form 24 different αβ heterodimers with differentspecificities for various ECM cell-adhesive proteins. Ligands forvarious integrins include fibronectin, collagen, laminin, von Willebrandfactor, osteopontin, thrombospondin, and vitronectin, which are allcomponents of the ECM. Certain integrins can also bind to solubleligands such as fibrinogen or to other adhesion molecules on adjacentcells. Integrins are known to exist in distinct activation states thatexhibit different affinities for ligand. Recognition of soluble ligandsby integrins strictly depends on specific changes in receptorconformation. This provides a molecular switch that controls the abilityof cells to aggregate in an integrin dependent manner and to arrestunder the dynamic flow conditions of the vasculature. This mechanism iswell established for leukocytes and platelets that circulate within theblood stream in a resting state while expressing non-activatedintegrins. Upon stimulation through proinflammatory or prothromboticagonists, these cell types promptly respond with a number of molecularchanges including the switch of key integrins, β2 integrins forleucocytes and αvβ3 for platelets, from “resting” to “activated”conformations. This enables these cell types to arrest within thevasculature, promoting cell cohesion and leading to thrombus formation.

It has been demonstrated that a metastatic subset of human breast cancercells expresses integrin αvβ3 in a constitutively activated form. Thisaberrant expression of αvβ3 plays a role in metastasis of breast canceras well as prostate cancer, melanoma, and neuroblastic tumors. Theactivated receptor strongly promotes cancer cell migration and enablesthe cells to arrest under blood flow conditions. In this way, activationof αvβ3 endows metastatic cells with key properties likely to becritical for successful dissemination and colonization of target organs.Tumor cells that have successfully entered a target organ may furtherutilize αvβ3 to thrive in the new environment, as αvβ3 matrixinteractions can promote cell survival and proliferation. For example,αvβ3 binding to osteopontin promotes malignancy and elevated levels ofosteopontin correlate with a poor prognosis in breast cancer.

For these reasons, and for its established role in angiogenesis, theαvβ3 integrin is one of the most widely studied integrins. Antagonistsof this molecule have significant potential for use in targeted drugdelivery. One approach that has been used to target αvβ3 integrin usesthe high binding specificity to αvβ3 of peptides containing theArg-Gly-Asp (RGD) sequence. This tripeptide, naturally present inextracellular matrix proteins, is the primary binding site of the αvβ3integrin. However, RGD based reporter probes are problematic due to fastblood clearance, high kidney and liver uptake and fast tumor washout.Chemical modification of cyclised RGD peptides has been shown toincrease their stability and valency. These modified peptides are thencoupled to radio-isotopes and used either for tumor imaging or toinhibit tumor growth.

Integrin αvβ3 is one of the most well characterized integrinheterodimers and is one of several heterodimers that have beenimplicated in tumor-induced angiogenesis. While sparingly expressed inmature blood vessels, αvβ3 is significantly up-regulated duringangiogenesis in vivo. The expression of αvβ3 correlates withaggressiveness of disease in breast and cervical cancer as well as inmalignant melanoma. Recent studies suggest that αvβ3 may be useful as adiagnostic or prognostic indicator for some tumors. Integrin αvβ3 isparticularly attractive as a therapeutic target due to its relativelylimited cellular distribution. It is not generally expressed onepithelial cells, and minimally expressed on other cell types.Furthermore, αvβ3 antagonists, including both cyclic RGD peptides andmonoclonal antibodies, significantly inhibit cytokine-inducedangiogenesis and the growth of solid tumor on the chick chorioallantoicmembrane.

Another integrin heterodimer, αvβ5, is more widely expressed onmalignant tumor cells and is likely involved in VEGF-mediatedangiogenesis. It has been shown that αvβ3 and αvβ5 promote angiogenesisvia distinct pathways: αvβ3 through bFGF and TNF-α, and αvβ5 throughVEGF and TGF-α. It has also been shown that inhibition of Src kinase canblock VEGF-induced, but not FGF2-induced, angiogenesis. These resultsstrongly imply that FGF2 and VEGF activate different angiogenic pathwaysthat require αvβ3 and αvβ5, respectively.

Integrins have also been implicated in tumor metastasis. Metastasis isthe primary cause of morbidity and mortality in cancer. Malignantprogression of melanoma, glioma, ovarian, and breast cancer have allbeen strongly linked with the expression of the integrin αvβ3 and insome cases with αvβ5. More recently, it has been shown that activationof integrin αvβ3 plays a significant role in metastasis in human breastcancer. A very strong correlation between expression of αvβ3 and breastcancer metastasis has been noted where normal breast epithelia are αvβ3negative and approximately 50% of invasive lobular carcinomas and nearlyall bone metastases in breast cancer express αvβ3. Antagonism of αvβ3with a cyclic peptide has been shown to synergize withradioimmunotherapy in studies involving breast cancer xenografts.

Angiogenesis, the formation of new blood vessels from existing ones, isessential to may physiological and pathological processes. Normally,angiogenesis is tightly regulated by pro- and anti-angiogenic factors,but in the case of diseases such as cancer, ocular neovascular disease,arthritis and psoriasis, the process can go awry. The association ofangiogenesis with disease has made the discovery of anti-angiogeniccompound attractive. The most promising phage derived anti-angiogenicpeptide described to date, developed by Amgen, neutralizes theangiogenic cytokine Ang2.

While the VEGFs and their receptors have been among the most extensivelytargeted molecules in the angiogenesis field, preclinical effortstargeting the more recently discovered angiopoietin-Tie2 pathway areunderway. Both protein families involve ligand receptor interactions,and both include members whose functions are largely restrictedpostnatally to endothelial cells and some hematopoietic stem celllineages. Tie-2 is a receptor tyrosine kinase with four known ligands,angiopoietin-1 (Ang1) through angiopoietin-4 (Ang4), the best studiedbeing Ang1 and Ang2. Ang1 stimulates phosphorylation of Tie2 and theAng2 interaction with Tie2 has been shown to both antagonize and agonizeTie2 receptor phosphorylation. Elevated Ang2 expression at sites ofnormal and pathological postnatal angiogenesis circumstantially impliesa proangiogenic role for Ang2. Vessel-selective Ang2 inductionassociated with angiogenesis has been demonstrated in diseases includingcancer. In patients with colon carcinoma, Ang2 is expressed ubiquitouslyin tumor epithelium, whereas expression of Ang1 in tumor epithelium wasshown to be rare. The net gain of Ang2 activity has been suggested to bean initiating factor for tumor angiogenesis.

Other fusion proteins directed towards cellular receptors are underclinical evaluation. Herceptin (Trastuzumab), developed by Genentech, isa recombinant humanized monoclonal antibody directed against theextracellular domain of the human epidermal tyrosine kinase receptor 2(HER2 or ErbB2). The HER2 gene is overexpressed in 25% of invasivebreast cancers, and is associated with poor prognosis and alteredsensitivity to chemotherapeutic agents. Herceptin blocks theproliferation of ErbB2-overexpressing breast cancers, and is currentlythe only ErbB2 targeted antibody therapy approved by the FDA for thetreatment of ErbB2 over-expressing metastatic breast cancer (MBC). Innormal adult cells, few ErbB2 molecules exist at the cell surface˜20,000 per cell, so few heterodimers are formed and growth signals arerelatively weak and controllable. When ErbB2 is overexpressed, ˜500,000per cell, multiple ErbB2 heterodimers are formed and cell signaling isstronger, resulting in enhanced responsiveness to growth factors andmalignant growth. This explains why ErbB2 overexpression is an indicatorof poor prognosis in breast tumors and may be predictive of response totreatment.

ErbB2 is a promising and validated target for breast cancer, where it isfound both in primary tumor and metastatic sites. Herceptin inducesrapid removal of ErbB2 from the cell surface, thereby reducing itsavailability to heterodimerize and promote growth. Mechanisms of actionof Herceptin observed in experimental in vitro and in vivo modelsinclude inhibition of proteolysis of ErbB2's extracellular domain,disruption of downstream signaling pathways such asphosphatidylinositiol 3-kinase (P13K) and mitogen-activated proteinkinase (MAPK) cascades, GI cell-cycle arrest, inhibition of DNA repair,suppression of angiogenesis and induction of antibody dependent cellularcytotoxicity (ADCC). The majority of patients with metastatic breastcancer who initially respond to Herceptin, however, demonstrate diseaseprogression within one year of treatment initiation.

Another target cellular receptor is type 1 insulin-like growth factor-1receptor (IGF-1R), IGF-1R is a receptor-tyrosine kinase that plays acritical role in signaling cell survival and proliferation. The IGFsystem is frequently deregulated in cancer cells by the establishment ofautocrine loops involving IGF-I or -II and/or IGF-1R overexpression.Moreover, epidemiological studies have suggested a link between elevatedIGF levels and the development of major human cancers, such as breast,colon, lung and prostate cancer. Expression of IGFs and their cognatereceptors has been correlated with disease stage, reduced survival,development of metastases and tumor de-differentiation.

Besides IGF-1R, epidermal growth factor receptor (EGFR) has also beenimplicated in the tumorigenesis of numerous cancers. Effective tumorinhibition has been achieved both experimentally and clinically with anumber of strategies that antagonize either receptor activity. Becauseof the redundancy of growth signaling pathways in tumor cells,inhibition of one receptor function (e.g. EGFR) could be effectivelycompensated by up-regulation of other growth factor receptor (e.g.IGF-1R)-mediated pathways. For example, a recent study has shown thatmalignant glioma cell lines expressing equivalent EGFR had significantlydifferent sensitivity to EGFR inhibition depending on their capabilityof activating IGF-1R and its downstream signaling pathways. Otherstudies have also demonstrated that overexpression and/or activation ofIGF-1R in tumor cells might contribute to their resistance tochemotherapeutic agents, radiation, or antibody therapy such asHerceptin. And consequently, inhibition of IGF-1R signaling has resultedin increased sensitivity of tumor cells to Herceptin.

EGFR is a receptor tyrosine kinase that is expressed on many normaltissues as well as neoplastic lesions of most organs. Overexpression ofEGFR or expression of mutant forms of EGFR has been observed in manytumors, particularly epithelial tumors, and is associated with poorclinical prognosis. Inhibition of signaling through this receptorinduces an anti-tumor effect. With the FDA approval of Cetuximab, alsoknown as Erbitux (a mouse/human chimeric antibody) in February of 2004,EGFR became an approved antibody drug target for the treatment ofmetastatic colorectal cancer. In March of 2006, Erbitux also receivedFDA approval for the treatment squamous cell carcinoma of the head andneck (SCCHN). More recently, Vectibix, a fully human antibody directedagainst EGFR, was approved for metastatic colorectal cancer. Neitherdrug is a stand-alone agent in colorectal cancer—they were approved asadd-ons to existing colorectal regimens. In colorectal cancer, Erbituxis given in combination with the drug irinotecan and Vectibix isadministered after disease progression on, or followingfluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapyregimens. Erbitux has been approved as a single agent in recurrent ormetastatic SCCHN only where prior platinum-based chemotherapy hasfailed. Advanced clinical trials which use these drugs to targetnon-small cell lung carcinoma are ongoing. The sequence of Erbitux orthe EGFR antibody, is well known in the art (see for example, Goldstein,et al., Clin. Cancer Res. 1:1311, 1995; U.S. Pat. No. 6,217,866), hereinincorporated by reference.

An obstacle in the utilization of a catalytic antibody for selectiveprodrug activation in cancer therapy has been systemic tumor targeting.The present invention describes an approach based on the adaptation oftarget binding peptides, or modular recognition domains (MRDs), whichare fused to full length antibodies that effectively target tumor cellsor soluble molecules while retaining the prodrug activation capabilityof the catalytic antibody. Since the MRDs are fused to the antibody soas not to significantly mitigate binding to the antibody's traditionalbinding site, the antibody's specificity remains intact after MRDaddition.

As noted in FIG. 2, MRDs, designated by triangles, circles, and squares,can be appended on any of the termini of either heavy or light chains ofa typical antibody. The first schematic represents a simple peptibodywith a peptide fused to the C-terminus of an Fc. This approach providedfor the preparation of bi-, tri-, tetra, and penta-specific antibodies.Display of a single MRD at each N- and C-termini of an IgG provides foroctavalent display of the MRD. As an alternative to the construction ofbi- and multifunctional antibodies through the combination of antibodyvariable domains, high-affinity peptides selected from phage displaylibraries or derived from natural ligands may offer a highly versatileand modular approach to the construction of multifunctional antibodiesthat retain both the binding and half-life advantages of traditionalantibodies. MRDs can also extend the binding capacity of non-catalyticantibodies, providing for an effective approach to extend the bindingfunctionality of antibodies, particularly for therapeutic purposes.

SUMMARY

The present invention is directed towards a full length antibodycomprising a modular recognition domain (MRD). Also embodied in thepresent invention are variants and derivitaves of such antibodiescomprising a MRD.

In one aspect, the antibody and the MRD are operably linked through alinker peptide. In one aspect, the linker peptide is between 2 to 20peptides long, or between 4 to 10 or about 4 to 15 peptides long. In oneaspect of the present invention, the linker peptide comprises thesequence GGGS (SEQ ID. NO.:1), the sequence SSGGGGSGGGGGGSS (SEQ ID.NO.: 2), or the sequence SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19). Otherlinkers containing a core sequence GGGS as shown in SEQ ID NO:1 areincluded herein wherein the linker peptide is from about 4-20 aminoacids.

According to another embodiment of the present invention, the MRD isoperably linked to the C-terminal end of the heavy chain of theantibody. In another aspect, the MRD is operably linked to theN-terminal end of the heavy chain of the antibody. In yet anotheraspect, the MRD is operably linked to the C-terminal end of the lightchain of the antibody. In another aspect, the MRD is operably linked tothe N-terminal end of the light chain of the antibody. In anotheraspect, two or more MRDs are operably linked to any terminal end of theantibody. In another aspect, two or more MRDs are operably linked to twoor more terminal ends of the antibody.

In one embodiment of the present invention, the target of the MRD is acellular antigen. In one embodiment of the present invention, the targetof the MRD is CD20.

In one embodiment of the present invention, the target of the MRD is anintegrin. In one aspect, the peptide sequence of the integrin targetingMRD is YCRGDCT (SEQ ID. NO.:3). In another aspect, the peptide sequenceof the integrin targeting MRD is PCRGDCL (SEQ ID. NO.:4). In yet anotheraspect, the peptide sequence of the integrin targeting MRD is TCRGDCY(SEQ ID. NO.:5). In another aspect, the peptide sequence of the integrintargeting MRD is LCRGDCF (SEQ ID. NO.:6).

In one embodiment of the present invention, the target of the MRD is anangiogenic cytokine. In one aspect, the peptide sequence of theangiogenic cytokine targeting MRD is MGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQID. NO.:7). In another aspect, the peptide sequence of the angiogeniccytokine targeting MRD is MGAQTNFMPMDNDELLLYEQFILQQGLE (SEQ ID. NO.:8).In yet another aspect, the peptide sequence of the angiogenic cytokinetargeting MRD is MGAQTNFMPMDATETRLYEQFILQQGLE (SEQ ID. NO.:9). Inanother aspect, the peptide sequence of the angiogenic cytokinetargeting MRD is AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE(SEQ ID. NO.:10). In another aspect, the peptide sequence of theangiogenic cytokine targeting MRD is MGAQTNFMPMDNDELLNYEQFILQQGLE (SEQID. NO.: 11). In another aspect, the peptide sequence of the angiogeniccytokine targeting MRD is PXDNDXLLNY (SEQ ID. NO.: 12), where X is oneof the 20 naturally-occurring amino acids. In another embodiment, thetargeting MRD peptide has the core sequence MGAQTNFMPMDXn (SEQ IDNO:56), wherein X is any amino acid and n is from about 0 to 15.

In another embodiment, the targeting MRD peptide contains a coresequence selected from:

XnEFAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:22);XnELAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:25);XnEFSPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:28);XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:31); andXn AQQEECEX₁X₂PWTCEHMXn where n is from about 0 to 50 amino acidresidues and X, X₁ and X₂ are any amino acid (SEQ ID NO:57).

Exemplary peptides containing such core peptides include for example:

(SEQ ID NO: 21) AQQEECEFAPWTCEHM; (SEQ ID NO: 23)AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCE HMLE; (SEQ ID NO: 24)AQQEECELAPWTCEHM; (SEQ ID NO: 26)AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE HMLE; (SEQ ID NO: 27)AQQEECEFSPWTCEHM; (SEQ ID NO: 29)AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCE HMLE 2xConFS;(SEQ ID NO: 30) AQQEECELEPWTCEHM; (SEQ ID NO: 32)AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCE HMLE; (SEQ ID NO: 33)AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE HMLE; (SEQ ID NO: 34)AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCE HMLE; and(SEQ ID. NO.: 10) AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE.

In one embodiment of the present invention, the target of the MRD isErbB2. In one embodiment of the present invention, the target of the MRDis ErbB3. In one embodiment of the present invention, the target of theMRD is tumor-associated surface antigen epithelial cell adhesionmolecule (Ep-CAM).

In one embodiment of the present invention, the target of the MRD isVEGF. In one aspect, the peptide sequence of the VEGF targeting MRD isVEPNCDIHVMWEWECFERL (SEQ ID. NO.:13).

In one embodiment of the present invention, the target of the MRD is aninsulin-like growth factor-I receptor. In one aspect, the peptidesequence of the insulin-like growth factor-I receptor targeting MRD isSFYSCLESLVNGPAEKSRGQWDGCRKK (SEQ ID NO:14). Other illustrative IGF-1Rtargeting MRDs include, for example, a peptide with the formulaNFYQCIX₁X₂LX₃X4X₅PAEKSRGQWQECRTGG (SEQ ID NO:58), wherein X₁ is E or D;X₂ is any amino acid; X₃ is any amino acid; X₄ is any amino acid and X₅is any amino acid.

Illustrative peptides that contain the formula include:

NFYQCIEMLASHPAEKSRGQWQECRTGG; (SEQ ID NO: 35)NFYQCIEQLALRPAEKSRGQWQECRTGG; (SEQ ID NO: 36)NFYQCIDLLMAYPAEKSRGQWQECRTGG; (SEQ ID NO: 37)NFYQCIERLVTGPAEKSRGQWQECRTGG; (SEQ ID NO: 38)NFYQCIEYLAMKPAEKSRGQWQECRTGG; (SEQ ID NO: 39)NFYQCIEALQSRPAEKSRGQWQECRTGG; (SEQ ID NO: 40)NFYQCIEALSRSPAEKSRGQWQECRTGG; (SEQ ID NO: 41)NFYQCIEHLSGSPAEKSRGQWQECRTG; (SEQ ID NO: 42)NFYQCIESLAGGPAEKSRGQWQECRTG; (SEQ ID NO: 43)NFYQCIEALVGVPAEKSRGQWQECRTG; (SEQ ID NO: 44)NFYQCIEMLSLPPAEKSRGQWQECRTG; (SEQ ID NO: 45)NFYQCIEVFWGRPAEKSRGQWQECRTG; (SEQ ID NO: 46)NFYQCIEQLSSGPAEKSRGQWQECRTG; (SEQ ID NO: 47)NFYQCIELLSARPAEKSRGQWAECRAG; (SEQ ID NO: 48) andNFYQCIEALARTPAEKSRGQWVECRAP. (SEQ ID NO: 49)

In one embodiment of the present invention, the target of the MRD is atumor antigen.

In one embodiment of the present invention, the target of the MRD is anepidermal growth factor receptor (EGFR). In one embodiment of thepresent invention, the target of the MRD is an angiogenic factor. In oneembodiment of the present invention, the target of the MRD is anangiogenic receptor.

In one embodiment of the present invention, the MRD is a vascular homingpeptide. In one aspect, the peptide sequence of the vascular homingpeptide is ACDCRGDCFCG (SEQ ID. NO:15).

In one embodiment of the present invention, the target of the MRD is anerve growth factor. In one of the present invention, the antibody bindsto a cell surface antigen.

In one embodiment of the present invention, the antibody or MRD binds toEGFR, ErbB2, ErbB3, ErbB4, CD20, insulin-like growth factor-I receptor,or prostate specific membrane antigen. In one aspect, the peptidesequence of the EGFR targeting MRD isVDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQA PK (SEQ ID NO:16). In one aspect, the peptide sequence of the EGFR targeting MRD isVDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQ APK (SEQ ID NO:17). In one aspect of the present invention, the peptide sequence ofErbB2 targeting MRD isVDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQA PK (SEQ ID NO:18).

In one embodiment of the present invention, the antibody binds to anangiogenic factor.

In one embodiment of the present invention, the antibody binds to anangiogenic receptor.

The present invention also relates to an isolated polynucleotidecomprising a nucleotide sequence of the antibody. In one aspect of thepresent invention, a vector comprises the polynucleotide. In yet anotheraspect, the polynucleotide is operatively linked with a regulatorysequence that controls expression on the polynucleotide. In one aspect,a host cell comprises the polynucleotide or progeny.

The present invention also relates to a method of treating a disease asubject in need thereof is provided, the method comprising administeringan antibody comprising an MRD. In one aspect, the disease is cancer. Inanother aspect, undesired angiogenesis in inhibited. In yet anotheraspect, angiogenesis is modulated. In yet another aspect, tumor growthis inhibited. In another embodiment, a method of treatment comprisingadministering an additional therapeutic agent along with an antibodycomprising an MRD is described.

The present invention also relates to a method of making a full lengthantibody comprising a MRD is described. In one aspect, the MRD isderived from a phage display library. IN another aspect, the MRD isderived from natural ligands.

In one embodiment of the present invention, the antibody is chimeric orhumanized.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the schematic representation of different designs oftetravalent IgG-like BsAbs.

FIG. 2A shows a typical peptibody as C-terminal fusion with Fc.

FIG. 2B shows an antibody with a C-terminal MRD fusion with the lightchain of the antibody.

FIG. 2C shows an antibody with an N-terminal MRD fusion with the lightchain of the antibody.

FIG. 2D shows an antibody with unique MRD peptides fused to eachterminus of the antibody.

FIG. 3 depicts the results of an ELISA in which integrin and Ang2 werebound by an anti-integrin antibody fused to a ang-2 targeting MRD.

FIG. 4 depicts the results of an ELISA in which integrin and Ang2 werebound by an anti-integrin antibody fused to a ang-2 targeting MRD.

FIG. 5 depicts the results of an ELISA in which an anti-ErbB2 antibodywas fused to an MRD which targeted Ang2.

FIG. 6 depicts the results of an ELISA in which an Ang2 targeting MRDwas fused to a hepatocyte growth factor receptor binding antibody.

FIG. 7 depicts the results of an ELISA in which an integrin targetingMRD was fused to an ErbB2 binding antibody.

FIG. 8 depicts the results of an ELISA in which an integrin targetingMRD was fused to an hepatocyte growth factor receptor binding antibody.

FIG. 9 depicts the results of an ELISA in which an insulin-like growthfactor-I receptor targeting MRD was fused to an ErbB2 binding antibody.

FIG. 10 depicts the results of an ELISA in which a VEGF-targeting MRDwas fused to an ErbB2 binding antibody.

FIG. 11 depicts the results of an ELISA in which an integrin targetingMRD was fused to a catalytic antibody.

FIG. 12 depicts the results of an ELISA in which an Ang-2-targeting MRDwas fused to a catalytic antibody.

FIG. 13 depicts the results of an ELISA in which an integrin and Ang-2targeting MRD was fused to an ErbB2 binding antibody.

FIG. 14 depicts the results of an ELISA in which an integrin targetingMRD was fused to an ErbB2-binding antibody.

FIG. 15 depicts the results of an ELISA in which an integrin, Ang-2, orinsulin-like growth factor-I receptor-targeting MRD was fused to anErbB2 or hepatocyte growth factor receptor-binding antibody with a shortlinker peptide.

FIG. 16 depicts the results of an ELISA in which an integrin, Ang-2, orinsulin-like growth factor-I receptor-targeting MRD was fused to anErbB2 or hepatocyte growth factor receptor-binding antibody with a longlinker peptide.

DETAILED DESCRIPTION OF THE INVENTION

The term “antibody” used herein to refer to intact immunoglobulinmolecules and includes polyclonal and monoclonal antibodies, chimeric,single chain, and humanized antibodies. An intact antibody comprises atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH₁, CH₂ andCH₃. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, C_(L). The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxyl-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen.

A “dual-specific antibody” is used herein to refer to an immunoglobulinmolecule which contain dual-variable-domain immunoglobins, where thedual-variable-domain can be engineered from any two monoclonalantibodies.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of a heavy and light chain variable and hypervariableregions that specifically binds (immunoreacts with) an antigen. The term“immunoreact” in its various forms means specific binding between anantigenic determinant-containing molecule and a molecule containing anantibody combining site such as a whole antibody molecule or a portionthereof.

The term “peptibody” refers to a peptide or polypeptide which comprisesless than a complete, intact antibody.

The term “naturally occurring” when used in connection with biologicalmaterials such as a nucleic acid molecules, polypeptides, host cells,and the like refers to those which are found in nature and not modifiedby a human being.

“Monoclonal antibody” refers to a population of antibody molecules thatcontain only one species of antibody combining site capable ofimmunoreacting with a particular epitope. A monoclonal antibody thustypically displays a single binding affinity for any epitope with whichit immunoreacts. A monoclonal antibody may therefore contain an antibodymolecule having a plurality of antibody combining sites, eachimmunospecific for a different epitope, e.g., a bispecific monoclonalantibody.

A “modular recognition domain” (MRD) or “target binding peptide” is amolecule, such as a protein, glycoprotein and the like, that canspecifically (non-randomly) bind to a target molecule. The amino acidsequence of a MRD site can tolerate some degree of variability and stillretain a degree of capacity to bind the target molecule. Furthermore,changes in the sequence can result in changes in the binding specificityand in the binding constant between a preselected target molecule andthe binding site.

“Cell surface receptor” refers to molecules and complexes of moleculescapable of receiving a signal and the transmission of such a signalacross the plasma membrane of a cell. An example of a cell surfacereceptor of the present invention is an activated integrin receptor, forexample, an activated αvβ3 integrin receptor on a metastatic cell.

The “target binding site” or “target site” is any known, or yet to bedescribed, amino acid sequence having the ability to selectively bind apreselected agent. Exemplary reference target sites are derived from theRGD-dependent integrin ligands, namely fibronectin, fibrinogen,vitronectin, von Willebrand factor and the like, from cellular receptorssuch as VEGF, ErbB2, vascular homing peptide or angiogenic cytokines,from protein hormones receptors such as insulin-like growth factor-Ireceptor, epidermal growth factor receptor and the like, and from tumorantigens.

The term “protein” is defined as a biological polymer comprising unitsderived from amino acids linked via peptide bonds; a protein can becomposed of two or more chains.

A “fusion polypeptide” is a polypeptide comprised of at least twopolypeptides and optionally a linking sequence to operatively link thetwo polypeptides into one continuous polypeptide. The two polypeptideslinked in a fusion polypeptide are typically derived from twoindependent sources, and therefore a fusion polypeptide comprises twolinked polypeptides not normally found linked in nature.

The term “linker” refers to a peptide located between the antibody andthe MRD. Linkers can have from about 2 to 20 amino acids, usually 4 to15 amino acids.

“Target cell” refers to any cell in a subject (e.g., a human or animal)that can be targeted by the antibody comprising an MRD of the invention.The target cell can be a cell expressing or overexpressing the targetbinding site, such as activated integrin receptor.

“Patient,” “subject,” “animal” or “mammal” are used interchangeably andrefer to mammals such as human patients and non-human primates, as wellas experimental animals such as rabbits, rats, and mice, and otheranimals. Animals include all vertebrates, e.g., mammals and non-mammals,such as sheep, dogs, cows, chickens, amphibians, and reptiles.

“Treating” or “treatment” includes the administration of the antibodycomprising an MRD of the present invention to prevent or delay the onsetof the symptoms, complications, or biochemical indicia of a disease,alleviating the symptoms or arresting or inhibiting further developmentof the disease, condition, or disorder. Treatment can be prophylactic(to prevent or delay the onset of the disease, or to prevent themanifestation of clinical or subclinical symptoms thereof) ortherapeutic suppression or alleviation of symptoms after themanifestation of the disease. Treatment can be with the antibody-MRDcomposition alone, or it can be used in combination with an additionaltherapeutic agent.

As used herein, the terms “pharmaceutically acceptable,” or“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a human without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

“Modulate,” means adjustment or regulation of amplitude, frequency,degree, or activity. In another related aspect, such modulation may bepositively modulated (e.g., an increase in frequency, degree, oractivity) or negatively modulated (e.g., a decrease in frequency,degree, or activity).

“Cancer,” “tumor,” or “malignancy” are used as synonymous terms andrefer to any of a number of diseases that are characterized byuncontrolled, abnormal proliferation of cells, the ability of affectedcells to spread locally or through the bloodstream and lymphatic systemto other parts of the body (metastasize) as well as any of a number ofcharacteristic structural and/or molecular features. A “cancerous,”“tumor,” or “malignant cell” is understood as a cell having specificstructural properties, lacking differentiation and being capable ofinvasion and metastasis. Examples of cancers are, breast, lung, brain,bone, liver, kidney, colon, head and neck, ovarian, hematopoietic (e.g.,leukemia), and prostate cancer.

“Humanized antibody” or “chimeric antibody” includes antibodies in whichCDR sequences derived from the germline of another mammalian species,such as a mouse, have been grafted onto human framework sequences.

The present invention describes an approach based on the adaptation oftarget binding peptides or modular recognition domains (MRDs) as fusionsto catalytic or non-catalytic antibodies that provide for effectivetargeting of tumor cells or soluble molecules while leaving the prodrugactivation capability of the catalytic antibody intact. MRDs can alsoextend the binding capacity of non-catalytic antibodies providing for aneffective approach to extend the binding functionality of antibodies,particularly for therapeutic purposes.

One aspect of the present invention relates to development of afull-length antibody comprising a modular recognition domain (MRD). Theinteraction between a protein ligand and its target receptor site oftentakes place at a relatively large interface. However, only a few keyresidues at the interface contribute to most of the binding. Thus,molecules of peptide length (generally 2 to 60 amino acids) can bind tothe receptor protein of a given large protein ligand. It is contemplatedthat MRDs of the present invention contain a peptide sequence that bindto target sites of interests and are about 2 to 60 amino acids.

The role of integrins such as αvβ3 and αvβ5 as tumor-associated markershas been well documented. A recent study of 25 permanent human celllines established from advanced ovarian cancer demonstrated that alllines were positive for αvβ5 expression and many were positive for αvβ3expression. Studies have also shown that αvβ3 and αvβ5 is highlyexpressed on malignant human cervical tumor tissues. Integrins have alsodemonstrated therapeutic effects in animal models of Kaposi's sarcoma,melanoma, and breast cancer.

A number of integrin αvβ3 and αvβ5 antagonists are in clinicaldevelopment. These include cyclic RGD peptides and synthetic smallmolecule ROD mimetics. Two antibody-based integrin antagonists arecurrently in clinical trials for the treatment of cancer. The first isVitaxin, the humanized form of the murine anti-human αvβ3 antibodyLM609. A dose-escalating phase I study in cancer patients demonstratedthat it was safe for use in humans. Another antibody in clinical trialsis CNT095, a fully human mAb that recognizes αv integrins. A Phase Istudy of CNT095 in patients with a variety of solid tumors has shownthat it is well tolerated. Cilengitide, a peptide antagonist of αvβ3 andαvβ5, has also proven safe in phase I trials. Furthermore, there hasbeen numerous drug targeting and imaging studies based on the use ofligands for these receptors. These preclinical and clinical observationsdemonstrate the importance of targeting αvβ3 and αvβ5 and studiesinvolving the use of antibodies in this strategy have consistentlyreported that targeting through these integrins is safe.

An example of an integrin-binding MRD is an RGD tripeptide-containingbinding site, and is exemplary of the general methods described herein.Ligands having the RGD motif as a minimum recognition domain are wellknown, a partial list of which includes, with the corresponding integrintarget in parenthesis, fibronectin (α3β1, α5β1, αvβ1, αIIbβ3, αvβ3, andα3β1) fibrinogen (αMβ2 and αIIbβ1) von Willebrand factor (αIIbβ3 andαvβ3), and vitronectin (αIIbβ3, αvβ3 and αvβ5).

Examples of RGD containing targeting MRDs useful in the presentinvention have amino acid residue sequences shown below:

YCRGDCT (SEQ ID. NO.: 3) PCRGDCL (SEQ ID. NO.: 4) TCRGDCY(SEQ ID. NO.: 5) LCRGDCF (SEQ ID. NO.: 6)

A MRD that mimics a non-RGD-dependent binding site on an integrinreceptor and having the target binding specificity of a high affinityligand that recognizes the selected integrin is also contemplated in thepresent invention.

Angiogenesis is essential to many physiological and pathologicalprocesses. Ang2 has been shown to act as a proangiogenic molecule.Administration of Ang2-selective inhibitors is sufficient to suppressboth tumor angiogenesis and corneal angiogenesis. Therefore, Ang2inhibition alone or in combination with inhibition of other angiogenicfactors such as VEGF may represent an effective antiangiogenic strategyfor treating patients with solid tumors.

It is contemplated that MRDs useful in the present invention includethose that bind to angiogenic receptors, angiogenic factors, and/orAng-2. Examples of angiogenic cytokine targeting MRD sequences arelisted below:

(SEQ ID. NO.: 7) MGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID. NO.: 8)MGAQTNFMPMDNDELLLYEQFILQQGLE (SEQ ID. NO.: 9)MGAQTNFMPMDATETRLYEQFILQQGLE (SEQ ID. NO.: 10)AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCE HMLE (2xCon4)(SEQ ID. NO.: 11) MGAQTNFMPMDNDELLNYEQFILQQGLE (SEQ ID. NO.: 12)PXDNDXLLNY where X is one of the 20 naturally-occurring  amino acids(SEQ ID NO: 20) MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMPMDNDELLLY (SEQ ID. NO.: 10)AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCE HMLE (SEQ ID NO: 21)AQQEECEFAPWTCEHM ConFA (SEQ ID NO: 22) core nEFAPWTnwhere n is from about 0 to 50 amino acid  residues (SEQ ID NO: 23)AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCE HMLE 2xConFA(SEQ ID NO: 24) AQQEECELAPWTCEHM ConLA (SEQ ID NO: 25)XnELAPWTXn where n is from about 0 to 50 amino acid residues and X is any amino acid (SEQ ID NO: 26)AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE HMLE 2xConLA(SEQ ID NO: 27) AQQEECEFSPWTCEHM ConFS (SEQ ID NO: 28)XnEFSPWTXn where n is from about 0 to 50 amino acid residues and X is any amino acid (SEQ ID NO: 29)AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCE HMLE 2xConFS(SEQ ID NO: 30) AQQEECELEPWTCEHM ConLE (SEQ ID NO: 31)XnELEPWTXn where n is from about 0 to 50  amino acid residues andwherein X is any amino acid (SEQ ID NO: 32)AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCE HMLE 2xConLE

It should be understood that such peptides can be present in dimmers,trimers or other multimers either homologous or heterologous in nature.For example, one can dimerize identical Con-based sequences such as in2xConFA to provide a homologous dimer, or the Con peptides can be mixedsuch that ConFA is combined with ConLA to create ConFA-LA heterodimerwith the sequence:

(SEQ ID NO: 33) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE HMLE.

Another heterodimer is ConFA combined with ConFS to create ConFA-FS withthe sequence:

(SEQ ID NO: 34) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCE HMLE.

One of skill in the art, given the teachings herein, will appreciatethat other such combinations will create functional Ang2 binding MRDs asdescribed herein.

In one aspect, the invention includes a peptide having the sequence:

NFYQCIX₁X₂LX₃X₄X₅PAEKSRGQWQECRTGG (SEQ ID NO:58), wherein X₁ is E or D;X₂ is any amino acid; X₃ is any amino acid; X₄ is any amino acid and X₅is any amino acid.

The invention also includes peptides having a core sequence selectedfrom:

XnEFAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:22);XnELAPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:25);XnEFSPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:28);XnELEPWTXn where n is from about 0 to 50 amino acid residues (SEQ ID NO:31); orXn AQQEECEX₁X₂PWTCEHMXn where n is from about 0 to 50 amino acidresidues and X, X₁ and X₂ are any amino acid (SEQ ID NO:57).

Phage display selections and structural studies of VEGF neutralizingpeptides in complex with VEGF have been reported. These studies haverevealed that peptide v114 (VEPNCDIHVMWEWECFERL) (SEQ ID. NO.: 13) isVEGF specific, binds VEGF with 0.2 affinity, and neutralizesVEGF-induced proliferation of Human Umbilical Vein Endothelial Cells(HUVEC). Since VEGF is a homodimer, the peptide occupies two identicalsites at either end of the VEGF homodimer. An antibody containing an MRDthat targets VEGF is contemplated in the present invention. Anti-VEGFantibodies can be found for example in Cancer Research 57, 4593-4599,October 1997; J Biol Chem 281:10 6625, 2006, herein incorporated byreference.

Insulin-like growth factor-I receptor-specific MRDs can be used in thepresent invention. One example of an MRD sequence that targets theinsulin-like growth factor-I receptor is SFYSCLESLVNGPAEKSRGQWDGCRKK(SEQ ID NO.: 14).

Additional IGF-1R MRDs include the following:

NFYQCIEMLASHPAEKSRGQWQECRTGG (SEQ ID NO: 35)NFYQCIEQLALRPAEKSRGQWQECRTGG (SEQ ID NO: 36)NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID NO: 37)NFYQCIERLVTGPAEKSRGQWQECRTGG (SEQ ID NO: 38)NFYQCIEYLAMKPAEKSRGQWQECRTGG (SEQ ID NO: 39)NFYQCIEALQSRPAEKSRGQWQECRTGG (SEQ ID NO: 40)NFYQCIEALSRSPAEKSRGQWQECRTGG (SEQ ID NO: 41) NFYQCIEHLSGSPAEKSRGQWQECRTG(SEQ ID NO: 42) NFYQCIESLAGGPAEKSRGQWQECRTG (SEQ ID NO: 43)NFYQCIEALVGVPAEKSRGQWQECRTG (SEQ ID NO: 44) NFYQCIEMLSLPPAEKSRGQWQECRTG(SEQ ID NO: 45) NFYQCIEVFWGRPAEKSRGQWQECRTG (SEQ ID NO: 46)NFYQCIEQLSSGPAEKSRGQWQECRTG (SEQ ID NO: 47) NFYQCIELLSARPAEKSRGQWAECRAG(SEQ ID NO: 48) NFYQCIEALARTPAEKSRGQWVECRAP (SEQ ID NO: 49)

A number of studies have characterized the efficacy of linking thevascular homing peptide to other proteins like IL-12 or drugs to directtheir delivery in live animals. As such, vascular homing MRDs arecontemplated for use in the present invention. One example of an MRDsequence that is a vascular homing peptide is ACDCRGDCFCG (SEQ ID NO.:15).

Numerous other target binding sites are contemplated by the presentinvention, including epidermal growth factor receptor (EGFR), CD20,tumor antigens, ErbB2, ErbB3, ErbB4, insulin-like growth factor-Ireceptor, nerve growth factor (NGR), hepatocyte growth factor receptor,and tumor-associated surface antigen epithelial cell adhesion molecule(Ep-CAM). MRDs can be directed towards these target binding sites.

Examples of MRD sequences that bind to EGFR are listed below:

(SEQ ID. NO.: 16) VDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK. (SEQ ID. NO.: 17)VDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKK LNDAQAPK.

An example of an MRD sequence that bind to ErbB2 is listed below:

(SEQ ID. NO.: 18) VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK.

The sequence of the MRD can be determined several ways. MRD sequencescan be derived from natural ligands or known sequences that bind to aspecific target binding site can be used. Additionally, phage displaytechnology has emerged as a powerful method in identifying peptideswhich bind to target receptors. In peptide phage display libraries,random peptide sequences can be displayed by fusion with coat proteinsof filamentous phage. The methods for elucidating binding sites onpolypeptides using phage display vectors has been previously described,in particular in WO 94/18221. The methods generally involve the use of afilamentous phage (phagemid) surface expression vector system forcloning and expressing polypeptides that bind to the pre-selected targetsite of interest.

The methods of the present invention for preparing MRDs involve the useof phage display vectors for their particular advantage of providing ameans to screen a very large population of expressed display proteinsand thereby locate one or more specific clones that code for a desiredtarget binding reactivity. Once the sequence of the MRD has beenelucidated, the peptides may be prepared by any of the methods disclosedin the art.

Variants and derivatives of the MRDs are included within the scope ofthe present invention. Included within variants are insertional,deletional, and substitutional variants as well as variants that includeMRDs presented here with additional amino acids at the N- and/orC-terminus, including from about 0 to 50, 0 to 40, 0 to 30, 0 to 20amino acids and the like. It is understood that a particular MRD of thepresent invention may contain one, two, or all three types of variants.Insertional and substitutional variants may contain natural amino acids,unconventional amino acids, or both.

It is contemplated that catalytic and non-catalytic antibodies can beused in the present invention. Antibody 38C2 is an antibody-secretinghybridoma, and has been previously described in WO 97/21803. 38C2contains an antibody combining site that catalyzes the aldol additionreaction between an aliphatic donor and an aldehyde acceptor. In asyngeneic mouse model of neuroblastoma, systemic administration of anetoposide prodrug and intra-tumor injection of Ab 38C2 inhibited tumorgrowth.

Other antibodies of interest to this invention include A33 bindingantibodies. Human A33 antigen is a transmembrane glycoprotein of the Igsuperfamily. The function of the human A33 antigen in normal andmalignant colon tissue is not yet known, however, several properties ofthe A33 antigen suggest that it is a promising target for immunotherapyof colon cancer. These properties include (i) the highly restrictedexpression pattern of the A33 antigen, (ii) the expression of largeamounts of the A33 antigen on colon cancer cells, (iii) the absence ofsecreted or shed A33 antigen, and (iv) the fact that upon binding ofantibody A33 to the A33 antigen, antibody A33 is internalized andsequestered in vesicles, and (v) the targeting of antibody A33 to A33antigen expressing colon cancer in preliminary clinical studies. Fusionof a MRD directed toward A33 to a catalytic or non-catalytic antibodywould increase the therapeutic efficacy of A33 targeting antibodies.

The present invention also contemplates the preparation of mono-, bi-,tri-, tetra-, and penta-specific antibodies. It is contemplated that theantibodies used in the present invention may be prepared by any methodknown in the art.

In the antibody-MRD fusion molecules prepared according to the presentinvention, the MRD may be attached to an antibody through the peptide'sN-terminus or C-terminus. The MRD may be attached to the antibody at theC-terminal end of the heavy chain of the antibody, the N-terminal end ofthe heavy chain of the antibody, the C-terminal end of the light chainof the antibody, or the N-terminal end of the light chain of theantibody. The MRD may be attached to the antibody directly, or attachedthrough an optional linker peptide, which can be between 2 to 20peptides long. The linker peptide can contain a short linker peptidewith the sequence GGGS (SEQ ID. NO.:1), a medium linker peptide with thesequence SSGGGGSGGGGGGSS (SEQ ID. NO.:2), or a long linker peptide withthe sequence SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19). The present inventionalso provides for two or more MRDs which are linked to any terminal endof the antibody. It is also contemplated that two or more MRDs can bedirectly attached or attached through a linker peptide to two or moreterminal ends of the antibody. The multiple MRDs can target the sametarget binding site, or two or more different target binding sites.Additional peptide sequences may be added to enhance the in vivostability of the MRD.

The antibody-MRD fusion molecules can be encoded by a polynucleoticecomprising a nucleotide sequence. A vector can contain thepolynucleotide sequence. The polynucleotide sequence can also be linkedwith a regulatory sequence that controls expression of thepolynucleotide in a host cell. A host cell, or its progeny, can containthe polynucleotide encoding the antibody-MRD fusion molecule.

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with at least one species of antibodycomprising an MRD as described herein, dissolved or dispersed therein asan active ingredient. In a preferred embodiment, the therapeuticcomposition is not immunogenic when administered to a human patient fortherapeutic purposes.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as sterile injectableseither as liquid solutions or suspensions, aqueous or non-aqueous,however, solid forms suitable for solution, or suspensions, in liquidprior to use can also be prepared. The preparation can also beemulsified. Thus, an antibody—MRD containing composition can take theform of solutions, suspensions, tablets, capsules, sustained releaseformulations or powders, or other compositional forms.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, propylene glycol,polyethylene glycol, and other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water.

Exemplary of such additional liquid phases are glycerin, vegetable oilssuch as cottonseed oil, organic esters such as ethyl oleate, andwater-oil emulsions.

A therapeutic composition contains an antibody comprising a MRD of thepresent invention, typically in an amount of at least 0.1 weight percentof antibody per weight of total therapeutic composition. A weightpercent is a ratio by weight of antibody to total composition. Thus, forexample, 0.1 weight percent is 0.1 grams of antibody-MRD per 100 gramsof total composition.

An antibody-containing therapeutic composition typically contains about10 microgram (ug) per milliliter (ml) to about 100 milligrams (mg) perml of antibody as active ingredient per volume of composition, and morepreferably contains about 1 mg/ml to about 10 mg/ml (i.e., about 0.1 to1 weight percent).

A therapeutic composition in another embodiment contains a polypeptideof the present invention, typically in an amount of at least 0.1 weightpercent of polypeptide per weight of total therapeutic composition. Aweight percent is a ratio by weight of polypeptide to total composition.Thus, for example, 0.1 weight percent is 0.1 grams of polypeptide per100 grams of total composition.

Preferably, an polypeptide-containing therapeutic composition typicallycontains about 10 microgram (ug) per milliliter (ml) to about 100milligrams (mg) per ml of polypeptide as active ingredient per volume ofcomposition, and more preferably contains about 1 mg/ml to about 10mg/ml (i.e., about 0.1 to 1 weight percent).

In view of the benefit of using humanized or chimeric antibodies in vivoin human patients, the presently described antibody-MRD molecules areparticularly well suited for in vivo use as a therapeutic reagent. Themethod comprises administering to the patient a therapeuticallyeffective amount of a physiologically tolerable composition containingan antibody comprising a MRD of the invention.

The dosage ranges for the administration of the antibody comprising aMRD of the invention are those large enough to produce the desiredeffect in which the disease symptoms mediated by the target molecule areameliorated. The dosage should not be so large as to cause adverse sideeffects, such as hyperviscosity syndromes, pulmonary edema, congestiveheart failure, and the like. Generally, the dosage will vary with theage, condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any complication.

A therapeutically effective amount of an antibody comprising a MRD ofthis invention is typically an amount of antibody such that whenadministered in a physiologically tolerable composition is sufficient toachieve a plasma concentration of from about 0.1 microgram (ug) permilliliter (ml) to about 100 ug/ml, preferably from about 1 ug/ml toabout 5 ug/ml, and usually about 5 ug/ml. Stated differently, the dosagecan vary from about 0.1 mg/kg to about 300 mg/kg, preferably from about0.2 mg/kg to about 200 mg/kg, most preferably from about 0.5 mg/kg toabout 20 mg/kg, in one or more dose administrations daily, for one orseveral days.

The antibody comprising a MRD of the invention can be administeredparenterally by injection or by gradual infusion over time. Although thetarget molecule can typically be accessed in the body by systemicadministration and therefore most often treated by intravenousadministration of therapeutic compositions, other tissues and deliverymeans are contemplated where there is a likelihood that the tissuetargeted contains the target molecule. Thus, antibodies comprising a MRDof the invention can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, transdermally, and can bedelivered by peristaltic means.

The therapeutic compositions containing a human monoclonal antibody or apolypeptide of this invention are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLES Example 1 Integrin Targeting Antibody-MRD Molecules

Novel antibody-MRD fusion molecules were prepared by fusion of anintegrin αvβ3-targeting peptides to catalytic antibody 38C2. Fusions atthe N-termini and C-termini of the light chain and the C-termini of theheavy chain were most effective. Using flow cytometry, the antibodyconjugates were shown to bind efficiently to integrin αvβ3-expressinghuman breast cancer cells. The antibody conjugates also retained theretro-aldol activity of their parental catalytic antibody 38C2, asmeasured by methodol and doxorubicin prodrug activation. Thisdemonstrates that cell targeting and catalytic antibody capability canbe efficiently combined for selective chemotherapy.

Example 2 Angiogenic Cytokine Targeting Antibody-MRD Molecules

Angiogenic cytokine targeting antibody—MRD fusion molecules wereconstructed. The antibody used was 38C2, which was fused with a MRDcontaining the 2xCon4 peptide(AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID. NO.:10)). The MRD peptide was fused to either the N- or C-terminus of thelight chain and the C-terminus of the heavy chain. Similar results werefound with the other Ang-2 MRD peptides. Additional Ang-2 MRD peptidesinclude:

LM-2x-32 (SEQ ID NO: 20)MGAQTNFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFM PMDNDELLLY(SEQ ID. NO.: 10) AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (2xCon4) (SEQ ID NO: 21) AQQEECEFAPWTCEHM ConFA (SEQ ID NO: 22)core XnEFAPWTXn where n is from about 0 to 50 amino acid residues(SEQ ID NO: 23) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCEHMLE 2xConFA (SEQ ID NO: 24) AQQEECELAPWTCEHM ConLA (SEQ ID NO: 25)XnELAPWTXn where n is from about 0 to 50 amino acid residues(SEQ ID NO: 26) AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCEHMLE 2xConLA (SEQ ID NO: 27) AQQEECEFSPWTCEHM ConFS (SEQ ID NO: 28)XnEFSPWTXn where n is from about 0 to 50 amino acid residues(SEQ ID NO: 29) AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCEHMLE 2xConFS (SEQ ID NO: 30) AQQEECELEPWTCEHM ConLE (SEQ ID NO: 31)XnELEPWTXn where n is from about 0 to 50 amino acid residues(SEQ ID NO: 32) AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCEHMLE 2xConLE.

It should be understood that such peptides can be present in dimmers,trimers or other multimers either homologous or heterologous in nature.For example, one can dimerize identical Con-based sequences such as in2xConFA to provide a homologous dimer, or the Con peptides can be mixedsuch that ConFA is combined with ConLA to create ConFA-LA heterodimerwith the sequence:

(SEQ ID NO: 33) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE HMLE.

Another illustrative heterodimer is ConFA combined with ConFS to createConFA-FS with the sequence:

(SEQ ID NO: 34) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCE HMLE.

One of skill in the art, given the teachings herein, will appreciatethat other such combinations will create functional Ang2 binding MRDs asdescribed herein.

Example 3 Antibody-MRD Fusions with Non-Catalytic Antibodies

A humanized mouse monoclonal antibody, LM609, directed towards humanintegrin αvβ3 has been previously described. (Rader, C. et. al., 1998.Rader C, Cheresh D A, Barbas C F 3rd. Proc Natl Acad Sci USA. 1998 Jul.21; 95(15):8910-5).

A human non-catalytic monoclonal Ab, JC7U was fused to an anti-Ang2 MRDcontaining 2xCon4(AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID. NO.:10)) at either the N- or C-terminus of the light chain. 2xCon4(AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID. NO.:10)) was studied as an N-terminal fusion to the Kappa chain of theantibody (2xCon4-JC7U) and as a C-terminal fusion (JC7U-2xCon4). Bothfusions maintained integrin and Ang2 binding. As shown in the left panelof FIG. 3, both antibody constructs (2xCon4-JC7U and JC7U-2xCon4)specifically bound to recombinant Ang2 as demonstrated by ELISA studies.Binding to Ang2, however, is significantly higher with JC7U-2xCon4,which has the 2xCon4 (SEQ ID. NO.: 10) fusion at the C-terminus of thelight chain of the antibody. The right panel of FIG. 3 depicts thebinding of Ang2-JC7U and JC7U-Ang2 to integrin αvβ3. The results showthat fusion of 2xCon4 (SEQ ID. NO.: 10) to either the N- or the C-lightchain terminus does not affect mAb JC7U binding to integrin αvβ3. FIG. 4depicts another ELISA study using the same antibody-MRD fusionconstructs.

Example 4 Herceptin-MRD Fusion Molecules

Another example of MRD fusions to a non-catalytic antibody areHerceptin-MRD fusion constructs. The Herceptin-MRD fusions aremultifunctional, both small-molecule αv integrin antagonists and thechemically programmed integrin-targeting antibody show remarkableefficacy in preventing the breast cancer metastasis by interfering withαv-mediated cell adhesion and proliferation. MRD fusions containingHerceptin-2xCon4 (which targets ErbB2 and ang2) and Herceptin-V114(which targets ErbB2 and VEGF targeting) and Herceptin-RGD-4C-2xCon4(which targets ErbB2, ang2, and integrin targeting) are effective.

Example 5 VEGF Targeting Antibody-MRD Molecules

An antibody containing an MRD that targets VEGF was constructed. A MRDwhich targets v114 (SEQ ID. NO. 13) was fused at the N-terminus of thekappa chain of 38C2 and Herceptin using the long linker sequence (SEQID. NO. 2). Expression and testing of the resulting antibody-MRD fusionconstructs demonstrated strong VEGF binding.

Example 6 IGF-1R Targeting Antibody-MRD Molecules

Fusion of an MRD which targets the IGF-1R (SFYSCLESLVNGPAEKSRGQWDGCRKK(SEQ ID. NO.: 14)) to the N-terminus of the kappa chain of 38C2 andHerceptin using the long linker sequence as a connector was studied.Expression and testing of the resulting antibody-MRD fusion constructsdemonstrated strong IGF-1R binding. Additional clones showing highbinding to IGR-1R were also identified after several rounds ofmutagenesis and screening. The preferred sequences listed below show nosignificant or no binding affinity to the insulin receptor. (see Table2).

TABLE 1 Template for further mutagenesis: Rm2-2-218 GTGGAGTGCAGGGCGCCGVECRAP SEQ ID NO: 50, 51 Rm2-2-316 GCTGAGTGCAGGGCTGGG AECRAGSEQ ID NO: 52, 53 Rm2-2-319 CAGGAGTGCAGGACGGGG QECRTG SEQ ID NO: 54, 55

TABLE 2 SEQ ID Mutant Amino acid sequence Template NO: Rm4-31NFYQCIEMLASHPAEKSRGQWQECRTGG Rm2-2-319 35 Rm4-33NFYQCIEQLALRPAEKSRGQWQECRTGG Rm2-2-319 36 Rm4-39NFYQCIDLLMAYPAEKSRGQWQECRTGG Rm2-2-319 37 Rm4-310NFYQCIERLVTGPAEKSRGQWQECRTGG Rm2-2-319 38 Rm4-314NFYQCIEYLAMKPAEKSRGQWQECRTGG Rm2-2-319 39 Rm4-316NFYQCIEALQSRPAEKSRGQWQECRTGG Rm2-2-319 40 Rm4-319NFYQCIEALSRSPAEKSRGQWQECRTGG Rm2-2-319 41 Rm4-44NFYQCIEHLSGSPAEKSRGQWQECRTG Rm2-2-319 42 Rm4-45NFYQCIESLAGGPAEKSRGQWQECRTG Rm2-2-319 43 Rm4-46NFYQCIEALVGVPAEKSRGQWQECRTG Rm2-2-319 44 Rm4-49NFYQCIEMLSLPPAEKSRGQWQECRTG Rm2-2-319 45 Rm4-410NFYQCIEVFWGRPAEKSRGQWQECRTG Rm2-2-319 46 Rm4-411NFYQCIEQLSSGPAEKSRGQWQECRTG Rm2-2-319 47 Rm4-415NFYQCIELLSARPAEKSRGQWAECRAG Rm2-2-316 48 Rm4-417NFYQCIEALARTPAEKSRGQWVECRAP Rm2-2-218 49

Example 7 ErbB2 Binding, Ang-2-Targeting Antibody-MRD Molecules

An antibody was constructed which contains an MRD that targets Ang-2(L17) fused to the light chain of an antibody which binds to ErbB2.Either the short linker sequence, the long linker sequence, or the 4thloop in the light chain constant region was used as a linker. FIG. 5depicts the results of an ELISA using constructs containing anN-terminal fusion of Ang-2 targeting MRD with the ErbB2 antibody withthe short linker peptide (GGGS (SEQ ID NO.: 1)) (L17-sL-Her), aC-terminal fusion of Ang-2 targeting MRD with the ErbB2 antibody withthe short linker peptide (Her-sL-L17), a C-terminal fusion of Ang-2targeting MRD with the ErbB2 antibody with the 4th loop in the lightchain constant region (Her-lo-L17), or an N-terminal fusion of Ang-2targeting MRD with the ErbB2 antibody with the long linker peptide(SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19)) (L17-lL-Her). ErbB2 was bound withvarying degrees by all of the constructs. However, Ang-2 was bound onlyby Her-sL-L17 and L17-lL-Her.

Example 8 Hepatocyte Growth Factor Receptor Binding, Ang-2-TargetingAntibody-MRD Molecules

Fusion of an MRD which targets Ang-2 (L17) was made to either theN-terminus or C-terminus of the light chain of the Met antibody, whichbinds to hepatocyte growth factor receptor. Either the short linkersequence or the long linker sequence were used as a connector. FIG. 6depicts the results of an ELISA using constructs containing N-terminalfusion of Ang-2 targeting MRD with the Met antibody with the shortlinker peptide (GGGS (SEQ ID NO.: 1)) (L17-sL-Met), N-terminal fusion ofAng-2 targeting MRD with the Met antibody with the long linker peptide(SSGGGGSGGGGGGSSRSS (SEQ ID NO.: 19)) (L17-lL-Met), and C-terminalfusion of Ang-2 targeting MRD with the Met antibody with the long linkerpeptide (Met-iL-L17). Expression and testing of the resultingantibody-MRD fusion constructs demonstrated strong Ang-2 binding whenthe long linker peptide was used. Fusion of the Ang-2 targeting MRD tothe C-light chain terminus of the antibody resulted in slightly higherbinding to Ang-2 then fusion of the Ang-2 targeting to the N-light chainterminus of the antibody.

Example 9 ErbB2 Binding, Integrin-Targeting Antibody-MRD Molecules

An antibody was constructed which contains an MRD that targets integrinαvβ3 (RGD4C) fused to the light chain of an antibody Herceptin whichbinds to ErbB2 (Her). Either the short linker sequence, the long linkersequence, or the 4th loop in the light chain constant region was used asa linker. FIG. 7 depicts the results of an ELISA using constructscontaining an N-terminal fusion of integrin αvβ3 targeting MRD with theErbB2 antibody with the short linker peptide (GGGS (SEQ ID NO.: 1))(RGD4C-sL-Her), a C-terminal fusion of integrin αvβ3 targeting MRD withthe ErbB2 antibody with the short linker peptide (Her-sL-RGD4C), aC-terminal fusion of integrin αvβ3 targeting MRD with the ErbB2 antibodywith the 4th loop in the light chain constant region (Her-lo-RGD4C), oran N-terminal fusion of integrin αvβ3 targeting MRD with the ErbB2antibody with the long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO.:19)) (RGD4C-lL-Her). ErbB2 was bound with varying degrees by all of theconstructs. However, integrin αvβ3 was bound only by RGD4C-lL-Her.

Example 10 Hepatocyte Growth Factor Receptor Binding, Integrin-TargetingAntibody-MRD Molecules

An antibody was constructed which contains an MRD that targets integrinαvβ3 (RGD4C) fused to the light chain of an antibody which binds to thehepatocyte growth factor receptor (Met). Antibody-MRD constructscontaining the long linker sequence were used. FIG. 8 depicts theresults of an ELISA using constructs containing an N-terminal fusion ofintegrin αvβ3 targeting MRD with the hepatocyte growth factor receptorantibody (RGD4C-lL-Met), or a C-terminal fusion of integrin αvβ3targeting MRD with the hepatocyte growth factor receptor antibody(Met-lL-RGD4C). The RGD4C-lL-Met demonstrated strong integrin αvβ3binding.

Example 11 ErbB2 Binding, Insulin-like Growth Factor-IReceptor-Targeting Antibody-MRD Molecules

Antibodies were constructed which contains an MRD that targetsinsulin-like growth factor-I receptor (RP) fused to the light chain ofan antibody which binds to ErbB2 (Her). Either the short linker peptide,the long linker peptide, or the 4th loop in the light chain constantregion was used as a linker. (Carter et al., Proc Natl Acad Sci USA.1992 May 15; 89(10):4285-9.

PMID: 1350088 [PubMed—indexed for MEDLINE]; U.S. Pat. No. 5,677,171;ATCC Deposit 10463, all incorporated by reference herein). FIG. 9depicts the results of an ELISA using constructs containing anN-terminal fusion of insulin-like growth factor-I receptor targeting MRDwith the ErbB2 antibody with the short (RP-sL-Her), a C-terminal fusionof insulin-like growth factor-I receptor targeting MRD with the ErbB2antibody and the short linker peptide (Her-sL-RP), a C-terminal fusionof insulin-like growth factor-I receptor targeting MRD with the ErbB2antibody with the 4th loop in the light chain constant region(Her-lo-RP), an N-terminal fusion of insulin-like growth factor-Ireceptor targeting MRD with the ErbB2 antibody with the long linkerpeptide (RP-lL-Her), or a C-terminal fusion of insulin-like growthfactor-I receptor targeting MRD with the ErbB2 antibody with the longlinker peptide (Her-lL-RP). ErbB2 was bound with varying degrees by allof the constructs. Insulin-like growth factor-1 receptor was bound byRP-lL-Her.

Example 12 ErbB2 Binding, VEGF-Targeting Antibody-MRD Molecules

Fusion of an MRD which targets VEGF (V114) was made to the N-terminus ofthe light chain of a ErbB2-binding antibody (Her). A medium linkerpeptide (SSGGGGSGGGGGGSS (SEQ ID NO.: 2)) was used as a connector. FIG.10 depicts the results of an ELISA using a construct containing anN-terminal fusion of VEGF targeting MRD with the ErbB2-binding antibodywith the medium linker peptide (V114-mL-Her). Expression and testing ofthe resulting antibody-MRD fusion construct demonstrated strong VEGF andErbB2 binding.

Example 13 Integrin Targeting Antibody-MRD Molecules

Fusion of an MRD which targets integrin αvβ3 (RGD) to the N-terminus ofthe light chain of 38C2 using the medium linker peptide as a connectorwas studied. FIG. 11 demonstrates that expression and testing of theresulting antibody-MRD fusion construct had strong integrin αvβ3binding.

Example 14 Ang-2 Targeting Antibody-MRD Molecules

Fusion of an MRD which targets Ang-2 (L17) to the C-terminus of thelight chain of 38C2 using the short linker sequence as a connector wasstudied. FIG. 12 demonstrates that expression and testing of theresulting antibody-MRD fusion construct had strong Ang-2 binding.

Example 15 ErbB2 Binding, Integrin and Ang-2 Targeting Antibody-MRDMolecules

An MRD which targets integrin αvβ3 (RGD4C) was connected to theN-terminus of the light chain of an ErbB2 targeting antibody (Her) witha medium linker, and an Ang-2 (L17) targeting MRD was connected by ashort linker to the C-terminus of the same ErbB2 targeting antibody(RGD4C-mL-Her-sL-L17). FIG. 13 demonstrates that the resultingantibody-MRD fusion construct bound to integrin, Ang-2, and ErbB2.

Example 16 ErbB2 Binding, Integrin-Targeting Antibody-MRD Molecules

An antibody was constructed which contains an MRD that targets integrinαvβ3 (RGD4C) fused to the N-terminus of the heavy chain of an antibodywhich binds to ErbB2 (Her) using the medium linker as a connector(RGD4C-mL-her-heavy). FIG. 14 depicts the results of an ELISA using theconstruct. Both integrin and ErbB2 were bound by the construct.

Example 17 ErbB2 or Hepatocyte Growth Factor Receptor Binding, andIntegrin, Ang-2 or Insulin-Like Growth Factor-I Receptor-TargetingAntibody-MRD Molecules with the Short Linker Peptide

Antibody-MRD molecules were constructed which contain ErbB2 orhepatocyte growth factor receptor binding antibodies, and integrin αvβ3,Ang-2 or insulin-like growth factor-I receptor-targeting MRD regionswere linked with the short linker peptide to the light chain of theantibody. FIG. 15 depicts the results of an ELISA using constructscontaining an N-terminal fusion of Ang-2 targeting MRD fused to theErbB2 antibody (L17-sL-Her), an N-terminal fusion of integrin-targetingMRD with the ErbB2 antibody (RGD4C-sL-Her), an N-terminal fusion ofinsulin-like growth factor-I receptor targeting MRD with the ErbB2binding antibody (RP-sL-Her), a C-terminal fusion of Ang-2 targeting MRDwith the hepatocyte growth factor receptor binding antibody(L17-sL-Met), a C-terminal fusion of Ang-2 targeting MRD with the ErbB2binding antibody (Her-sL-L17), a C-terminal fusion of integrin targetingMRD with the ErbB2 binding antibody (Her-sL-RGD4C), or a C-terminalfusion of insulin-like growth factor-I receptor targeting MRD with theErbB2 binding antibody (Her-sL-RP). ErbB2 was bound with varying degreesby the antibody-MRD constructs, with the exception of the constructcontaining the hepatocyte growth factor receptor-binding antibody.Antigen was bound only by the Her-sL-L17 construct.

Example 18 ErbB2 or Hepatocyte Growth Factor Receptor Binding, andIntegrin, Ang-2 or Insulin-like Growth Factor-I Receptor-TargetingAntibody-MRD Molecules with the Long Linker Peptide

Antibody-MRD molecules were constructed which contain ErbB2 orhepatocyte growth factor receptor binding antibodies, and integrin αvβ3,Ang-2 or insulin-like growth factor-I receptor-targeting MRD regionslinked with the long linker peptide to the light chain of the antibody.FIG. 16 depicts the results of an ELISA using constructs containing anN-terminal fusion of Ang-2 targeting MRD fused to the ErbB2 antibody(L17-lL-Her), an N-terminal fusion of integrin-targeting MRD with theErbB2 antibody (RGD4C-lL-Her), an N-terminal fusion of insulin-likegrowth factor-I receptor-targeting MRD with the ErbB2 binding antibody(RP-lL-Her), a C-terminal fusion of Ang-2 targeting MRD with thehepatocyte growth factor receptor binding antibody (L17-lL-Met), aC-terminal fusion of integrin targeting MRD with the hepatocyte growthfactor receptor binding antibody (RGD4C-lL-Met), a C-terminal fusion ofAng-2 targeting MRD with the insulin-like growth factor-I receptorbinding antibody (Her-lL-RP), a C-terminal fusion of Ang-2 targeting MRDwith the hepatocyte growth factor receptor binding antibody (Met-lL-L17), or a C-terminal fusion of integrin targeting MRD with thehepatocyte growth factor receptor binding antibody (Met-lL-RGD4C). Asshown in FIG. 16, antibody-MRD fusions are effective to bind antigen andErbB2. Lu et al. J Biol. Chem. 2005 May 20; 280(20):19665-72. Epub 2005Mar. 9; Lu et al. J Biol. Chem. 2004 Jan. 23; 279(4):2856-65. Epub 2003Oct. 23,

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. An isolated full length antibody comprising a modular recognitiondomain (MRD).
 2. The antibody of claim 1, wherein the antibody and theMRD are operably linked through a linker peptide.
 3. The antibody ofclaim 2, wherein the linker peptide is between 2 to 20 peptides.
 4. Theantibody of claim 2, wherein the linker peptide is between 4 to 15peptides.
 5. The antibody of claim 2, wherein the linker peptidecomprises the sequence GGGS (SEQ ID NO:1) (SEQ ID. NO.: 1).
 6. Theantibody of claim 2, wherein the linker peptide comprises the sequenceSSGGGGSGGGGGGSS (SEQ ID NO:2) (SEQ ID. NO 2).
 7. The antibody of claim2, wherein the linker peptide comprises the sequence SSGGGGSGGGGGGSSRSS(SEQ ID NO:19) (SEQ ID NO 19).
 8. The antibody of claim 1, wherein theMRD is operably linked to the C-terminal end of the heavy chain of theantibody.
 9. The antibody of claim 1, wherein the MRD is operably linkedto the N-terminal end of the heavy chain of the antibody.
 10. Theantibody of claim 1, wherein the MRD is operably linked to theC-terminal end of the light chain of the antibody.
 11. The antibody ofclaim 1, wherein the MRD is operably linked to the N-terminal end of thelight chain of the antibody.
 12. The antibody of claim 1, wherein two ormore MRDs are operably linked to any terminal end of the antibody. 13.The antibody of claim 1, wherein there are two or more MRDs operablylinked to two or more terminal ends of the antibody.
 14. The antibody ofclaim 1, wherein the target of the MRD is an integrin.
 15. The antibodyof claim 14, wherein the integrin-targeting MRD comprises the sequenceYCRGDCT (SEQ ID NO:3) (SEQ ID. NO.: 3).
 16. The antibody of claim 14,wherein the integrin-targeting MRD comprises the sequence PCRGDCL (SEQID NO:4) (SEQ ID. NO.: 4)).
 17. The antibody of claim 14, wherein theintegrin-targeting MRD comprises the sequence TCRGDCY (SEQ ID NO:5) (SEQID. NO.: 5).
 18. The antibody of claim 14, wherein theintegrin-targeting MRD comprises the sequence LCRGDCF (SEQ ID NO:6) (SEQID. NO.: 6).
 19. The antibody of claim 15 wherein the target of the MRDis an angiogenic cytokine.
 20. The antibody of claim 19, wherein theangiogenic cytokine-targeting MRD comprises the sequenceMGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO:7) (SEQ ID. NO.: 7).
 21. Theantibody of claim 19, wherein the angiogenic cytokine-targeting MRDcomprises the sequence MGAQTNFMPMDNDELLLYEQFILQQGLE (SEQ ID NO:8) (SEQID. NO.: 8).
 22. The antibody of claim 19, wherein the angiogeniccytokine-targeting MRD comprises the sequenceMGAQTNFMPMDATETRLYEQFILQQGLE (SEQ ID NO:9) (SEQ ID. NO.: 9).
 23. Theantibody of claim 19, wherein the angiogenic cytokine-targeting MRD isselected from the group consisting of: (SEQ ID NO: 10) (SEQ ID. NO.: 10)AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCE HMLE; (SEQ ID NO: 21)AQQEECEFAPWTCEHM; (SEQ ID NO: 23)AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCE HMLE; (SEQ ID NO: 24)AQQEECELAPWTCEHM; (SEQ ID NO: 26)AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE HMLE; (SEQ ID NO: 27)AQQEECEFSPWTCEHM; (SEQ ID NO: 29)AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCE HM LE2xConFS;(SEQ ID NO: 30) AQQEECELEPWTCEHM; (SEQ ID NO: 32)AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCE HMLE; (SEQ ID NO: 33)AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE HMLE; and(SEQ ID NO: 34) AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCE HMLE.


24. The antibody of claim 19, wherein the angiogenic cytokine-targetingMRD comprises the sequence (SEQ ID NO: 11) (SEQ ID. NO.: 11)MGAQTNFMPMDNDELLNYEQFILQQGLE. 


25. The antibody of claim 19, wherein the angiogenic cytokine-targetingMRD comprises the sequence PXDNDXLLNY (SEQ ID NO:12) (SEQ ID. NO.: 12),where X is selected from one of the 20 naturally-occurring amino acids.26. The antibody of claim 1, wherein the target of the MRD is ErbB2. 27.The antibody of claim 1, wherein the target of the MRD is VEGF.
 28. Theantibody of claim 27, wherein the VEGF-targeting MRD comprises thesequence VEPNCDIHVMWEWECFERL (SEQ ID NO:13) (SEQ ID. NO.: 13).
 29. Theantibody of claim 1, wherein the target of the MRD is an insulin-likegrowth factor-I receptor.
 30. The antibody of claim 29, wherein theinsulin-like growth factor-I receptor-targeting MRD is selected from thegroup consisting of: SFYSCLESLVNGPAEKSRGQWDGCRKK; (SEQ ID NO: 14)(SEQ ID. NO.: 14) NFYQCIEMLASHPAEKSRGQWQECRTGG; (SEQ ID NO: 35)NFYQCIEQLALRPAEKSRGQWQECRTGG; (SEQ ID NO: 36) NFYQCIDLLMAYPAEKSRGQWQECRTGG; (SEQ ID NO: 37)NFYQCIERLVTGPAEKSRGQWQECRTGG; (SEQ ID NO: 38)NFYQCIEYLAMKPAEKSRGQWQECRTGG; (SEQ ID NO: 39)NFYQCIEALQSRPAEKSRGQWQECRTGG; (SEQ ID NO: 40)NFYQCIEALSRSPAEKSRGQWQECRTGG; (SEQ ID NO: 41)NFYQCIEHLSGSPAEKSRGQWQECRTG; (SEQ ID NO: 42)NFYQCIESLAGGPAEKSRGQWQECRTG; (SEQ ID NO: 43)NFYQCIEALVGVPAEKSRGQWQECRTG; (SEQ ID NO: 44)NFYQCIEMLSLPPAEKSRGQWQECRTG; (SEQ ID NO: 45)NFYQCIEVFWGRPAEKSRGQWQECRTG; (SEQ ID NO: 46)NFYQCIEQLSSGPAEKSRGQWQECRTG; (SEQ ID NO: 47)NFYQCIELLSARPAEKSRGQWAECRAG; (SEQ ID NO: 48) andNFYQCIEALARTPAEKSRGQWVECRAP. (SEQ ID NO: 49)


31. The antibody of claim 1, wherein the target of the MRD is a tumorantigen.
 32. The antibody of claim 1, wherein the target of the MRD isCD20.
 33. The antibody of claim 1, wherein the target of the MRD is anepidermal growth factor receptor (EGFR).
 34. The antibody of claim 33,wherein the EGFR-targeting MRD comprises the sequence (SEQ ID NO: 16)(SEQ ID. NO.: 16) VDNKFNKELEKAYNEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQA PK.


35. The antibody of claim 33, wherein the EGFR-targeting MRD comprisesthe sequence (SEQ ID NO: 17) (SEQ ID. NO.: 17)VDNKFNKEMWIAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKK LNDAQAPK.


36. The antibody of claim 1, wherein the target of the MRD is the ErbB2receptor.
 37. The antibody of claim 36, wherein the ErbB2receptor-targeting MRD comprises the sequence (SEQ ID NO: 18)(SEQ ID. NO.: 18) VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK.


38. The antibody of claim 1, wherein the target of the MRD is the ErbB3receptor.
 39. The antibody of claim 1, wherein the target of the MRD istumor-associated surface antigen epithelial cell adhesion molecule(Ep-CAM).
 40. The antibody of claim 1, wherein the target of the MRD isan angiogenic factor.
 41. The antibody of claim 40, wherein the antibodybinds to a cell surface antigen selected from the group consisting ofEGFR, ErbB2, ErbB3, ErbB4, CD20 insulin-like growth factor-I receptor,or prostate specific membrane antigen.
 42. The antibody of claim 1,wherein the target of the MRD is an angiogenic receptor.
 43. Theantibody of claim 42, wherein the antibody binds to a cell surfaceantigen selected from the group consisting of EGFR, ErbB2, ErbB3, ErbB4,CD20, insulin-like growth factor-I receptor, or prostate specificmembrane antigen.
 44. The antibody of claim 42, wherein the antibodybinds to an angiogenic factor.
 45. The antibody of claim 42, wherein theantibody binds to an angiogenic receptor.
 46. The antibody of claim 1,wherein the target of the MRD is a cell surface antigen.
 47. Theantibody of claim 46, wherein the antibody binds to a cell surfaceantigen.
 48. The antibody of claim 47, wherein the cell surface antigenis selected from the group consisting of EGFR, ErbB2, ErbB3, ErbB4,CD20, insulin-like growth factor-I receptor, or prostate specificmembrane antigen.
 49. The antibody of claim 46, wherein the antibodybinds to an angiogenic factor.
 50. The antibody of claim 46, wherein theantibody binds to an angiogenic receptor.
 51. The antibody of claim 1,wherein the MRD is a vascular homing peptide.
 52. The antibody of claim51, wherein the vascular homing peptide-targeting MRD comprises thesequence ACDCRGDCFCG (SEC) ID NO:15) (SEQ ID. NO.: 15).
 53. The antibodyof claim 1, wherein the MRD is a nerve growth factor (NGF).
 54. Anisolated polynucleotide comprising a nucleotide sequence of the antibodyof claim
 1. 55. A vector comprising the polynucleotide of claim
 54. 56.A vector comprising the polynucleotide of claim 54 in which thenucleotide sequence of the polynucleotide is operatively linked with aregulatory sequence that controls expression of the polynucleotide in ahost cell.
 57. A host cell comprising the vector of claim 55, or progenyof the cell.
 58. A method of treating a disease comprising administeringto a subject in need thereof, an antibody of claim
 1. 59. The method ofclaim 58, wherein the disease is cancer.
 60. The method of claim 58,wherein an additional therapeutic agent is administered to the subject.61. A method of inhibiting angiogenesis comprising administering to asubject in need thereof, an antibody of claim
 1. 62. The method of claim61, wherein an additional therapeutic agent is administered to thesubject.
 63. A method of modulating angiogenesis comprisingadministering to a subject in need thereof, an antibody of claim
 1. 64.The method of claim 63, wherein an additional therapeutic agent isadministered to the subject.
 65. A method of inhibiting tumor growthcomprising administering to a subject in need thereof, an antibody ofclaim
 1. 66. The method of claim 65, wherein an additional therapeuticagent is administered to the subject.
 67. A method for producing a fulllength antibody comprising one or more MRDs, said method comprisingselecting for a MRD using an MRD binding target, wherein the MRD isderived from a phage display library.
 68. A method for producing a fulllength antibody comprising one or more MRDs, said method comprisingselecting for a MRD using an MRD binding target, wherein the MRD isderived from natural ligands.
 69. The antibody of claim 1, wherein theantibody is a chimeric or humanized antibody.
 70. A peptide comprisingthe sequence NFYQCIX₁X₂LX₃X₄X₅PAEKSRGQWQECRTGG (SEQ ID NO:58), whereinX₁ is E or D X₂ is any amino acid; X₃ is any amino acid; X₄ is any aminoacid and X_(s) is any amino acid.
 71. A peptide of claim 70, selectedfrom the group consisting of: NFYQCIEMLASHPAEKSRGQWQECRTGG;(SEQ ID NO: 35) NFYQCIEQLALRPAEKSRGQWQECRTGG; (SEQ ID NO: 36)NFYQCIDLLMAYPAEKSRGQWQECRTGG; (SEQ ID NO: 37)NFYQCIERLVTGPAEKSRGQWQECRTGG; (SEQ ID NO: 38)NFYQCIEYLAMKPAEKSRGQWQECRTGG; (SEQ ID NO: 39)NFYQCIEALQSRPAEKSRGQWQECRTGG; (SEQ ID NO: 40)NFYQCIEALSRSPAEKSRGQWQECRTGG; (SEQ ID NO: 41)NFYQCIEHLSGSPAEKSRGQWQECRTG; (SEQ ID NO: 42)NFYQCIESLAGGPAEKSRGQWQECRTG; (SEQ ID NO: 43)NFYQCIEALVGVPAEKSRGQWQECRTG; (SEQ ID NO: 44)NFYQCIEMLSLPPAEKSRGQWQECRTG; (SEQ ID NO: 45)NFYQCIEVFWGRPAEKSRGQWQECRTG; (SEQ ID NO: 46) andNFYQCIEQLSSGPAEKSRGQWQECRTG. (SEQ ID NO: 47)


72. A peptide selected from (SEQ ID NO: 20)MGAQINFMPMDNDELLLYEQFILQQGLEGGSGSTASSGSGSSLGAQTNFM PMDNDELLLY;(SEQ ID NO: 48) NFYQCIELLSARPAEKSRGQWAECRAG; or (SEQ ID NO: 49)NFYQCIEALARTPAEKSRGQWVECRAP.


73. A peptide selected from the group consisting of: X_(n)EFAPWTX_(n)where n is from about 0 to 50 amino acid residues (SEQ ID NO: 22);X_(n)ELAPWTX_(n) where n is from about 0 to 50 amino acid residues (SEQID NO: 25); X_(n)EFSPWTX_(n) where n is from about 0 to 50 amino acidresidues (SEQ ID NO: 28); X_(n)ELEPWTX_(n) where n is from about 0 to 50amino acid residues (SEQ ID NO: 31); and Xn AQQEECEX_(I)X₂PWTCEHMX_(n)where n is from about 0 to 50 amino acid residues and X, X₁ and X₂ areany amino acid (SEQ ID NO:57).
 74. A peptide of claim 73, wherein thepeptide is selected from the group consisting of: (SEQ ID NO: 21)AQQEECEFAPWTCEHM; (SEQ ID NO: 23)AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFAPWTCE HMLE; (SEQ ID NO: 24)AQQEECELAPWTCEHM; (SEQ ID NO: 26)AQQEECELAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE HMLE; (SEQ ID NO: 27)AQQEECEFSPWTCEHM; (SEQ ID NO: 29)AQQEECEFSPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCE HM LE2xConFS;(SEQ ID NO: 30) AQQEECELEPWTCEHM; (SEQ ID NO: 32)AQQEECELEPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELEPWTCE HMLE; (SEQ ID NO: 33)AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECELAPWTCE HMLE; (SEQ ID NO: 34)AQQEECEFAPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEFSPWTCE HMLE; and(SEQ ID NO: 10) (SEQ ID. NO.: 10)AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCE HMLE.


75. An antigen-binding construct comprising a protein scaffold which islinked to one or more epitope-binding domains wherein theantigen-binding construct has at least two antigen binding sites atleast one of which is from an epitope binding domain and at least one ofwhich is from a paired VH/VL domain.
 76. An antigen-binding construct ofclaim 75 wherein the binding construct has specificity for more than oneantigen.
 77. An antigen-binding construct of claim 75 wherein theantigen-binding construct is capable of binding two or more antigensselected from VEGF, IGF-1R and EGFR.
 78. An antigen-binding construct ofclaim 75 wherein the antigen-binding construct is capable of bindingTNF.
 79. An antigen-binding construct of claim 75 wherein the scaffoldis an IgG scaffold.
 80. An antigen-binding construct of claim 75 whereinthe protein scaffold comprises a monovalent antibody.
 81. Anantigen-binding construct of claim 75 wherein the IgG scaffold comprisesall the domains of an antibody.
 82. An antigen-binding construct ofclaim 75 wherein at least one of the epitope binding domains is directlyattached to the Ig scaffold with a linker comprising from 1 to 20 aminoacids.
 83. An antigen-binding construct of claim 82 wherein at least oneof the epitope binding domains is directly attached to the Ig scaffoldwith a linker that is GGGS (SEQ ID NO:1).
 84. An antigen-bindingconstruct of claim 77 comprising an epitope binding domain attached tothe Ig scaffold at the N-terminus of the light chain.
 85. Anantigen-binding construct of claim 77 comprising an epitope bindingdomain attached to the Ig scaffold at the N-terminus of the heavy chain.86. An antigen-binding construct of claim 77 comprising an epitopebinding domain attached to the Ig scaffold at the C-terminus of thelight chain.
 87. An antigen-binding construct of claim 77 comprising anepitope binding domain attached to the Ig scaffold at the C-terminus ofthe heavy chain.
 88. An antigen-binding construct of claim 75 which has4 antigen binding sites and which is capable of binding 4 antigenssimultaneously.
 89. An antigen-binding construct of claim 75 for use inmedicine.
 90. An antigen-binding construct of claim 75 for use in themanufacture of a medicament for treating cancer or arthritis.
 91. Amethod of treating a patient suffering from cancer or arthritis,comprising administering a therapeutic amount of an antigen-bindingconstruct of claim
 75. 92. An antigen-binding construct of claim 75 forthe treatment of cancer or arthritis.
 93. A polynucleotide sequenceencoding a heavy chain of an antigen binding construct of claim
 75. 94.A polynucleotide encoding a light chain of an antigen binding constructof claim
 75. 95. A recombinant transformed or transfected host cellcomprising one or more polynucleotide sequences encoding a heavy chainand a light chain of an antigen binding construct of claim
 75. 96. Apharmaceutical composition comprising an antigen binding construct ofclaim 75 and a pharmaceutically acceptable carrier.
 97. An antibodyconstruct comprising an antibody which is linked to one or more MRDs,wherein the construct has at least two antigen binding sites at leastone of which is from an MRD and at least one of which is from theantibody combining site.
 98. The construct of claim 97, wherein theconstruct has specificity for more than one antigen.
 99. The constructof claim 97, wherein the construct is capable of binding two or moreantigens selected from VEGF, IGF-1R, and EGFR.
 100. The construct ofclaim 97, wherein the construct is capable of binding TNF.
 101. Theconstruct of claim 97, wherein the antibody is an IgG antibody.
 102. Theconstruct of claim 97 wherein the antibody comprises a monovalentantibody.
 103. The construct of claim 97, wherein at least one of theMRDs is directly attached to the antibody with a linker comprising from2 to 20 amino acids.
 104. The construct of claim 103, wherein at leastone MRD is directly attached to the antibody with a linker that is GGGS(SEQ ID NO:1).
 105. The construct of claim 99 comprising an MRD operablylinked to the antibody at the N-terminal end of the light chain. 106.The construct of claim 99, comprising an MRD operably linked to theantibody at the N-terminal end of the heavy chain.
 107. The construct ofclaim 99, comprising an MRD operably linked to the antibody at theC-terminal end of the light chain.
 108. The construct of claim 99,comprising an MRD operably linked to the C-terminal end of the heavychain.
 109. An antigen-binding construct of claim 97 which has 4 antigenbinding sites and which is capable of binding 4 antigens simultaneously.110. The construct of claim 97 for use in therapeutic purposes.
 111. Theconstruct of claim 97 for use in the manufacture of a medicament fortreating cancer or arthritis.
 112. A method of treating a patient withcancer or arthritis, comprising administering a therapeutic amount ofthe construct of claim
 97. 113. The construct of claim 97, for thetreatment of cancer or arthritis.
 114. A polynucleotide sequenceencoding the construct of claim
 97. 115. A host cell comprising thepolynucleotide of claim
 97. 116. A therapeutic composition comprisingthe construct of claim 97 and a physiologically tolerable carrier.